This invention relates to the field of protein chemistry, and more particularly, to the field of fibrinolytic enzymes.
A number of enzymes that influence blood coagulation have been isolated from various snake venoms. These enzymes can either promote or inhibit coagulation. Fibrinolytic activities have received special attention because of their possible therapeutic role.
U.S. Pat. No. 4,610,879 (the entire disclosure of which is hereby incorporated by reference) describes the isolation and purification of a fibrinolytic enzyme (fibrolase or Accfib) from the venom of Agkistrodon contortrix contortrix (Southern copperhead snake). This enzyme has been of great potential therapeutic interest in view of its direct fibrinolytic activity which is not readily inhibited by serum antiprotease when employed in vivo. Unlike urokinase and streptokinase, which operate by the conversion of plasminogen to the proteolytic enzyme plasmin, A.c.contortrix fibrolase is not a plasminogen activator. Plasmin degrades other plasma proteins, as well as fibrin clots. When plasmin forced by a plasminogen activator exceeds the capacity of the circulating plasmin inhibitor, alpha-2-antiplasmin, fibrinogen and other clotting factors will be depleted; this enhances the probability of hemorrhagic complications common after thrombolytic therapy. Fibrolase as isolated from A.c.contortrix does not exhibit any detectable plasminogen activator activity. In addition, A.c.contortrix fibrolase is free of the significant toxic side effects (such as hemolytic or hemorrhagic properties) associated with the crude venom from which it is derived. Therefore, A.c.contortrix fibrolase is being investigated as a promising agent for treatment of myocardial infarction, acute deep-vein thrombosis and pulmonary embolism.
The fibrinolytic enzyme from A.c.contortrix has been purified by conventional liquid chromatography methods, by high performance liquid chromatography (HPLC) and by isoelectric focusing, and characterized. This enzyme is a zinc-dependent metalloproteinase (one mole zinc per mole enzyme) with a molecular weight of 23,000. It readily cleaves the .alpha.- and A.alpha.-chain of fibrin and fibrinogen, respectively, between Lys.sup.413 -Leu.sup.414 without activating or degrading plasminogen or Protein C. Accfib has been shown to dissolve aged clots in the renal arteries and iliac veins of rabbits in vivo, and therefore may have significant clinical potential.
A non-hemorrhagic fibrinolytic enzyme, called atroxase, has been isolated from the venom of Crotalus atrox [Willis, T. W. and Tu, A. T., Biochemistry 27:4769-4777 (1988)]. This enzyme is a zinc-dependent metalloproteinase with a molecular weight similar to that of Accfib. Its in vivo activity has been studied in rats [Willis, T. W. et al., Thrombos. Res 53: 19-29 (1989)].
Unfortunately, in addition to its direct fibrinolytic properties, A.c.contortrix fibrolase also exhibits a significant degree of general proteolytic activity (as exemplified by azocaseinolytic activity). Thus, in spite of its potential utility in short-term administrations, the properties of the enzyme as a general protease suggest that high dosages or long-term administration thereof might result in the destruction of other systemic proteins through general proteolysis.
It is an object of the present invention to provide novel fibrinolytic agents with peptide bond cleavage specificities which differ from those of the heretofore known fibrinolytic agents, for use as therapeutic agents (for example, in the dissolution of blood clots) and as templates for the development of de novo agents.
It is a further object of the present invention to provide direct fibrinolytic agents with the desirable lack of observable systemic toxicity (as exemplified by the absence of hemorrhagic activity) of A.c.contortrix fibrolase but without the degree of proteolytic activity exhibited thereby.